EP3412288A1 - Vegf-hemmer zur verwendung zur vorbeugung und/oder behandlung von akne - Google Patents

Vegf-hemmer zur verwendung zur vorbeugung und/oder behandlung von akne Download PDF

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Publication number
EP3412288A1
EP3412288A1 EP17305679.7A EP17305679A EP3412288A1 EP 3412288 A1 EP3412288 A1 EP 3412288A1 EP 17305679 A EP17305679 A EP 17305679A EP 3412288 A1 EP3412288 A1 EP 3412288A1
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EP
European Patent Office
Prior art keywords
vegf
acne
inhibitor
cdc1
acnes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP17305679.7A
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English (en)
French (fr)
Inventor
Emmanuel Vial
Gilles Ouvry
Fériel HACINI-RACHINEL
Florent GINHOUX
Baptiste JANELA
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Galderma Research and Development SNC
Agency for Science Technology and Research Singapore
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Galderma Research and Development SNC
Agency for Science Technology and Research Singapore
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Application filed by Galderma Research and Development SNC, Agency for Science Technology and Research Singapore filed Critical Galderma Research and Development SNC
Priority to EP17305679.7A priority Critical patent/EP3412288A1/de
Priority to PCT/EP2018/065080 priority patent/WO2018224614A1/en
Publication of EP3412288A1 publication Critical patent/EP3412288A1/de
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention concerns the use of VEGF inhibitors for preventing and/or treating acne.
  • Acne is a common multi-factor pathology that attacks skin rich in sebaceous glands (face, shoulder area, arms and intertriginous areas). It is the most commonly occurring form of dermatosis. The following five pathogenic factors play a determining role in the development of acne:
  • acne conglobata acne keloid on the nape of the neck
  • acne medicamentosa recurrent miliary acne
  • acne necrotica acne necrotica
  • acne neonatorum premenstrual acne
  • occupational acne acne rosacea
  • senile acne solar acne and acne vulgaris.
  • Acne vulgaris also known as polymorphous juvenile acne, is the most common. It comprises four stages, but it is not necessary to pass through all the stages:
  • the various forms of acne described above can be treated with active agents, such as antiseborrhoeics and antiinfectives, for example benzoyl peroxide (in particular, the product Eclaran® marketed by Pierre Fabre); with retinoids, such as tretinoin (in particular, the product Retacnyl® marketed by Galderma) or isotretinoin (the product Roaccutane® marketed by Laboratoires Roche); or with naphthoic acid compounds, such as, in particular, 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid, commonly known as adapalene (the product Differine® marketed by Galderma).
  • active agents such as antiseborrhoeics and antiinfectives, for example benzoyl peroxide (in particular, the product Eclaran® marketed by Pierre Fabre); with retinoids, such as tretinoin (in particular, the product Re
  • Combinations of several local treatments (such as antibiotics, retinoids, peroxides or zinc) is also used in dermatology to increase the efficacy of the active ingredients and to reduce their toxicity, but the multiple application of various dermatological products can be quite laborious and demanding for the patient.
  • the present invention now offers a treatment for reaching this purpose, and offers an improved therapy for acne, preferably via the topical route.
  • the present invention aims to use VEGF inhibitors for preventing and/or treating acne. Indeed, targeting VEGF and inhibiting its expression, activity and/or signaling pathway, such as via the topical route (i.e. on skin), would allow controlling, and thereby reducing, the recruitment of neutrophils, thus preventing and/or decreasing the inflammatory response to P.acnes bacterium, and thereby the development of acne lesions.
  • the invention thus relates to a VEGF inhibitor for use for preventing and/or treating acne, preferably via the topical route.
  • the invention further concerns a method of preventing and/or treating acne, comprising administering to a patient in need thereof a therapeutically effective amount of a VEGF inhibitor.
  • acne it is understood, all acne forms especially simple acne, comedogenic acne, papulopustular acne, papulocomedonic acne, nodulocystic acne, acne conglobata, cheloid acne of the nape of the neck, recurrent miliary acne, necrotic acne, neonatal acne, occupational acne, acne rosacea, senile acne, solar acne, medication-related acne and acne vulgaris.
  • the invention is intended for preventing and/or treating acne vulgaris.
  • the VEGF inhibitor is used for preventing and/or treating acne, by reducing the recruitment of neutrophils.
  • the VEGF inhibitor prevents and/or decreases the inflammatory response to P.acnes bacterium.
  • the VEGF inhibitor prevents and/or decreases the development of acne lesions.
  • the VEGF inhibitor prevents and/or decreases VEGF-A-signalling on neutrophils induced by cDC1, particularly by the subset of EpCAM + CD59 + Ly6D + cDC1.
  • VEGF-A-signalling on neutrophils it is meant VEGF-R1 expression on neutrophils.
  • the subset of EpCAM + CD59 + Ly6D + cDC1 is a subset of dendritic cells.
  • Dendritic cells are professional pathogen-sensing and antigen-presenting cells (APC) that are central to the initiation and regulation of immune responses (Schlitzer et al., 2015).
  • Conventional dendritic cells are innate immune cells.
  • the term cDC refers to all DCs other than plasmacytoid DCs.
  • cDC ontogenetically-distinct and functionally-specialized conventional DC
  • cDC1 identified in mice as expressing CD103 (integrin ⁇ E); and cDC2, expressing the CD11b (integrin ⁇ M), and SIRP ⁇ (Schlitzer and Ginhoux, 2014, Schlitzer et al., 2015, Guilliams et al., 2016).
  • LC Langerhans cells
  • MHCII skin cDC
  • CD207 langerin
  • CD24 CD24
  • CD11c macrophages
  • F4/80 macrophages
  • subset of EpCAM + CD59 + Ly6D + cDC1 it is meant the subset of cDC which may be present in skin and which express the surface markers CD103 (or integrin ⁇ E), EpCAM (epithelial cell adhesion molecule), CD59 (or MAC-inhibitory protein or membrane inhibitor of reactive lysis or MIRL) and Ly6D (or lymphocyte antigen 6 complex, locus D; or E48).
  • VEGF Vascular Endothelial Growth Factor
  • VEGFRs tyrosine kinase receptors
  • the VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like domains, a single transmembrane spanning region, and an intracellular portion containing a tyrosine-kinase domain.
  • VEGF-A binds to VEGFR-1 and VEGFR-2.
  • VEGF-C and VEGF-D but not VEGF-A, are ligands for a third receptor (VEGFR-3), which mediates lymphangiogenesis.
  • VEGF inhibitor any compound that inhibits or reduces VEGF biological activity.
  • the biological activity of VEGF depends on the amount of the VEGF nucleic acid (i.e. its expression level) and/or on the amount of the VEGF protein; on VEGF interaction with its target sequences, particularly its receptor(s) (called VEGFR); or on the activation of VEGFR. Therefore, the VEGF inhibitor may reduce or inhibit VEGF expression, or reduce or inhibit VEGF interaction ability with its target sequences, particularly its receptor(s), or reduce or inhibit the activation of VEGFR.
  • the VEGF inhibitor is a VEGF-A inhibitor.
  • VEGF expression refers to events modifying VEGF mRNA transcriptionally or post-transcriptionally, by cleavage and maturation, to provide a functional VEGF, notably any reaction which results in inhibition of VEGF mRNA processing; it also includes events modifying VEGF protein during translation, as well as post-translational modifications.
  • target sequence of VEGF according to the invention is a sequence to which VEGF specifically binds.
  • the target sequence is VEGF-R1, VEGF-R2 or VEGF-R3, more preferably VEGF-R1.
  • an “inhibitor of VEGF expression” refers to any compound that has a biological effect to inhibit the expression of a VEGF gene and/or the expression of a VEGF protein.
  • said inhibitor of VEGF gene expression is a siRNA, or an antisense oligonucleotide.
  • Small inhibitory RNAs siRNAs
  • Gene expression can be reduced with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • Inhibitors of VEGF for use in the invention may be based on antisense oligonucleotide (ODNs) constructs.
  • ODNs antisense oligonucleotide constructs.
  • Antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, would act to directly block the activity of VEGF by binding to VEGF mRNA and thus preventing binding leading to mRNA degradation.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the VEGF transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135 ; 6,566,131 ; 6,365,354 ; 6,410,323 ; 6,107,091 ; 6,046,321 ; and 5,981,732 ).
  • antisense oligonucleotides may be modified with phosphorothioate to prevent their in vivo hydrolysis by nucleases. Such modifications are well known in the art.
  • Antisense oligonucleotides useful as inhibitors of VEGF can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. They can also be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • the terms “inhibitor of the interaction” means preventing or reducing the direct or indirect association of one or more molecules, nucleic acids, peptides, proteins.
  • the term “inhibitor of the interaction between VEGF and its target sequences, particularly its receptor(s)” is a molecule which can prevent the interaction between VEGF and its target sequences, particularly its receptor(s), particularly VEGF-R1, by competition or by fixing to one of the molecules.
  • the VEGF inhibitor is an inhibitor of the interaction between VEGF and its receptor, preferably VEGF-R1, VEGF-R2 or VEGF-R3.
  • the receptor is VEGF-R1.
  • VEGF inhibitor is an antibody, and preferably bevacizumab, ranibizumab or ramucirumab.
  • the VEGF inhibitor is a molecule which can prevent the activation of VEGFR.
  • a VEGF inhibitor is called a VEGFR tyrosine kinase inhibitor.
  • the test for determining whether a compound is a VEGFR tyrosine kinase inhibitor is the VEGFR kinase assay, and notably the VEGF-R2 kinase assay:
  • VEGFR tyrosine kinase inhibitor is chosen from:
  • the VEGF inhibitor is advantageously formulated in a pharmaceutical composition, together with a pharmaceutically acceptable carrier.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • the pharmaceutical compositions of the invention can be formulated for a topical, oral, intraocular, intravenous, intramuscular or subcutaneous administration and the like. Preferably they are topical.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being topically applied.
  • the composition in particular the pharmaceutical composition, according to the invention, can be present in all galenic forms normally used for topical administration.
  • preparations may be mentioned in liquid, pasty or solid form and, more particularly, in the form of ointments, aqueous, aqueous-alcoholic or oily solutions, dispersions of the optionally two-phase lotion type, serum, aqueous, anhydrous or lipophilic gels, powders, soaked pads, syndets, wipes, sprays, foams, sticks, shampoos, compresses, washing bases, emulsions of liquid or semi-liquid consistency such as milk, obtained by dispersing a fatty phase in an aqueous phase (O/W) or inversely (W/O), a microemulsion, suspensions or emulsions of soft, semi-liquid or solid of white or colored
  • the physiologically acceptable medium may comprise various excipients.
  • excipient it is meant an inert substance typically used as a diluent or carrier for the VEGF inhibitor according to the invention.
  • Emulsions such as oil-in-water (O/W) or water-in-oil (W/O) systems, as well as a base (vehicle or support) for the topical formulation, may be chosen so as to ensure efficacy of the active ingredients and/or to avoid allergic and irritant reactions.
  • the compositions may comprise an emulsifier.
  • Non-limiting examples of emulsifiers useful in this regard include glycol esters, fatty acids, fatty alcohols, fatty acid esters of glycols, fatty esters, fatty ethers, glycerine esters, propylene glycol esters, polyethylene glycol fatty acid esters, fatty acid esters of polypropylene glycol, sorbitol esters, esters of sorbitan anhydrides, copolymers of carboxylic acids, glucose esters and ethers, ethoxylated ethers, ethoxylated alcohols, alkyl phosphates, polyoxyethylene phosphate ethers, fatty acid amides, acyl lactylates, soaps and mixtures thereof.
  • emulsifiers useful in the present compositions include polyethylene glycol-20 sorbitan monolaurate (polysorbate-20), polyethylene glycol, soybean sterol, steareth-2, steareth-20, steareth-21, ceteareth-20, glucose methyl ether distearate PPG-2, ceteth-10, polysorbate-80, cetyl phosphate, potassium cetyl phosphate, diethanolamine cetyl phosphate, polysorbate-60, glyceryl stearate, PEG-100 stearate, tragacanth gum and mixtures thereof.
  • the lotions useful in the present compositions may be suspensions of powdered material in an aqueous or alcoholic base.
  • Ointments are oleaginous compositions that contain little or no water (anhydrous).
  • the compositions can also be in the form of gels.
  • the compositions may comprise a gelling agent and/or a thickener.
  • Suitable gelling and/or thickening agents which may be useful in the present compositions include aqueous thickeners, such as neutral, anionic and cationic polymers, and mixtures thereof.
  • Examples of polymers which may be useful in the present compositions include carboxyvinyl polymers, such as carboxypolymethylene.
  • a preferred thickener is a carbomer, for example a Carbopol® polymer from Noveon Inc.
  • polymers useful in this regard include hydrophilic/hydrophobic graft copolymers, such as polymers formed as a mixture of polystyrene/microsponge/Carbopol®.
  • a polymer in this respect is a dimethyl acrylamide/acrylic acid/polystyrene ethyl methacrylate copolymer, for example a copolymer of the Pharmadur® brand as available from Polytherapeutics.
  • suitable thickeners include cellulosic polymers such as arabic gum, tragacanth gum, locust bean gum, guar gum, hydroxypropylguar, xanthan gum, cellulose gum, sclerotium gum, carrageenan gum, karaya gum, cellulose, rosin, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylhydroxyethylcellulose, cetylhydroxyethylcellulose, carboxymethylcellulose, corn starch, hydroxypropyl phosphate starch, PEG-150/alkoxy stearyl alcohol/SMDI copolymer, PEG-180/laureth-50/TMMG copolymer, acrylic acid/acrylamidomethylpropane sulfonic acid copolymer, acrylate/C10-30 acrylate copolymer, acrylate/beheneth-25 methacrylate copolymer, acrylate/
  • the pharmaceutical composition may be adapted for injection.
  • the compositions may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • 'treatment' includes therapeutic treatment
  • 'prevention' includes prophylactic or preventive treatment, wherein the object is to prevent or slow down the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • the term "treating” includes reducing, alleviating or inhibiting or eliminating the symptoms or progress of a disorder.
  • an effective amount preferably a therapeutically effective amount of the VEGF inhibitor of the invention is administered.
  • An "effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a "therapeutically effective amount" of a VEGF inhibitor of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the VEGF inhibitor, to elicit a desired therapeutic result.
  • a therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the VEGF inhibitor are outweighed by the therapeutically beneficial effects.
  • a therapeutically effective amount also encompasses an amount sufficient to confer benefit, e.g., clinical benefit.
  • C57BL/6 (C45.2 + ) mice were from the Biological Resource Center, Agency for Science, Technology and Research (A*STAR), Singapore.
  • Congenic C57BL/6 (C45.1 + ) mice were from Jackson Laboratory. Langerin DTR mice were provided by B. Malissen. Batf3 -/- and ZBTB46 -GFP BM were provided by K. Murphy.
  • HIF flox mice and IRF4 flox mice were crossed with CD11c cre + mice to give rise to congenic CD11c cre + HIF flox mice and CD11c cre + IRF4 flox mice respectively. All mice were bred and maintained in the Singapore Immunology Network animal facility before use at 7-10 weeks of age.
  • mice at 6-8 weeks of age were lethally irradiated with two doses of 600 rads, 5 hr apart, and then injected i.v. with 1 x 10 6 cells from femurs and tibias of Batf3 -/- , Zbtb46 gfp , and langerin DTR mice. All experiments and procedures were approved by the Institutional Animal Care and Use Committee of the Biological Resource Center (Agency for Science, Technology and Research, Singapore) in accordance with the guidelines of the Agri-Food and Veterinary Authority and the National Advisory Committee for Laboratory Animal Research of Singapore.
  • P. acnes strain ATCC 6919 was obtained from American Type Culture Collections and grown in an anaerobic chamber at 37°C for 5-6 days. A spectrophotometer reading OD 525 was used to identify the bacterial log growth phase.
  • Live P. acnes was intradermally injected (10 8 CFU in 20 ⁇ l of PBS) into the left ear of mice; while the right ear was injected with an equal volume of PBS.
  • DT was administered intraperitoneally (i.p.).
  • Recombinant mouse VEGF 164 protein 300 ng, R&S system
  • anti-vegfa (2 ⁇ g, Abcam) have been intradermally injected concomitantly with P. acnes. Ear thickness was measured using an electronic caliper (Mitutoyo).
  • Frozen skin samples embedded in OCT compound (Sakura Finetek) were sliced into 5- ⁇ m-thick sections and fixed with 1% paraformaldehyde (PFA) before staining with Mayer's hematoxylin and eosin (H&E) or picosirius red.
  • PFA paraformaldehyde
  • mice skin cells were isolated as described previously [ Ginhoux, F., M. P. Collin, et al. (2007). "Blood-derived dermal langerin+ dendritic cells survey the skin in the steady state.” J Exp Med 204(13): 3133-3146 ]. Briefly, mouse ears were split into dorsal and ventral halves and floated in RPMI-1640 medium (Sigma) containing 1 mg/ml dispase (Invitrogen) for 60 min at 37°C to allow separation of epidermal and dermal sheets.
  • the separated sheets were then cut into small pieces and incubated in RPMI containing 10% serum, Collagenase IV (0.2mg / ml, Roche) and DNAse I (20000U / ml, Roche) for 1 hr at 37°C. Cells were then passed through 19 G needle and filtered through a 100 ⁇ M cell strainer (BD Falcon) to obtain a homogenous cell suspension.
  • Multi-parameter analyses and sorting of labelled cell suspensions were performed on a LSR II and FACSAria II (Becton Dickinson, San Jose, USA) respectively. Data were analysed with FlowJo software (TreeStar). Fluorochrome- or biotin- conjugated monoclonal antibodies (mAbs) against the following molecules used: mouse IA/IE (M5/114.15.2) (BD Biosciences); Ly6G (1A8), CD64 (X54-5/7.1), EpCAM (G8.8) (Biolegend) and CD11c (N418), CD45 (30F11), CD45.1 (A20), CD45.2 (104), Ly6C (HK1.4), CD24 (M1/69), FC ⁇ RI (MAR-1), BST2 (eBio927), and CD11b (M1/70) (all from eBioscience).
  • the streptavidin-CF594 conjugate (562284) was from BD Biosciences.
  • cell suspensions from mouse ears were first depleted of T and B cells using antibodies against CD90 and CD19, respectively.
  • the remaining cells were placed in a U-bottom 96-well plate (BD Falcon, cat. no. 3077), washed once with 200 ⁇ L flow cytometry buffer (4% FBS, 2 mM EDTA, 0.05% Azide in 1 ⁇ PBS) and then stained with 100 ⁇ L of 200 ⁇ M cisplatin (Sigma-Aldrich, cat. no. 479306-1 G) for 5 min on ice to exclude dead cells.
  • Table 1 Table of antibodies used for mass cytometry analysis listing metal conjugate, antibody clone name, supplier Isotope tag Sequence Clone/Company Qdot (Cd112/114) CD19 6D5 (Invitrogen) Rh--103 Barcode Pd--104 Barcode Pd---105 Barcode Pd--106 Barcode Pd--108 Barcode Pd--110 Barcode Ln--113 Barcode Pr--141 CD45 30--F11 (Biolegend) Nd--142 MHC class II Y3P (BioXcell) Nd--143 B220 RA3.341/61 (BioXC
  • fluorophore-(phycoerythrin or allophycocyanin) or biotin-conjugated antibodies were used as primary antibodies followed by secondary staining with anti-fluorophore metal-conjugated antibodies (anti-APC, (Clone APC003, BioLegend cat. no. 408002, 5 ⁇ g/mL), anti-PE, (Clone PE001, BioLegend cat. no. 408102, 5 ⁇ g/mL)) or metal-conjugated streptavidin.
  • anti-APC anti-fluorophore metal-conjugated antibodies
  • anti-PE anti-PE
  • Clone PE001, BioLegend cat. no. 408102 5 ⁇ g/mL
  • streptavidin metal-conjugated streptavidin
  • DEG Differentially-expressed genes
  • Skin cDC1 were isolated after P. acnes infection by fluorescence-activated cell sorting, and were diluted to a final concentration of 400 cells per ⁇ l.
  • Cells were loaded onto C 1 integrated fluidic circuits (IFC) (5- to 10- ⁇ m chip) for capture, cell lysis, reverse transcription with oligo (dT) primers, and amplification of cDNA on a C1 Single-cell Auto Prep System according to the protocol of the manufacturer (Fluidigm). Reads were mapped against the mm10 reference genome with the Bowtie alignment program. The estimated expression values, in transcripts per million (TPM), for each gene annotated with gencode gene annotation version M4 was calculated using the RSEM program.
  • TPM transcripts per million
  • TPM values 0 in all cells were discarded. TPM values less than 1 were considered background, and log2 TPM was set to 0 as a conservative background cut-off. Positive TPM values then underwent log2 transformation. There were no outlier cells identified with the Singular Analysis Toolset with default parameters. Using the Singular Analysis Toolset, the inventors selected a subset of 8238 genes whose log2 TPM values were greater than 1 at both sample-average level and across all the cells, for all downstream analyses.
  • the PCA function using genes expressed in more than two cells with a non-zero variance, highlighted and ranked the genes with the most variability in expression across all the cells (data not shown); together with the hierarchical clustering function in the Singular Toolset (data not shown) this identified two subsets comprising 8 and 59 cells respectively.
  • the ANOVA function in the Singular Toolset identified genes that were significantly differentially expressed in these two cell subsets: 1796 genes were identified, with P values of ⁇ 0.05. Among these genes, 1556 were upregulated and 240 were downregulated in the cell subset comprising of 8 cells, compared to the larger 59-cell subset. Multiple-testing correction was done by the Benjamini-Hochberg method.
  • the sorting points into neighborhoods (SPIN) algorithm was applied to the top 100 genes with highest loadings associated with the first principal component of PCA and independently identified the two cell subsets comprising the same individual cells (data not shown).
  • Pathway enrichment analysis was performed using the IPA software on the 240 genes expressed more abundantly in the minor subset, with P values ⁇ 0.05 considered significant. Except for the Matlab implementation of SPIN, or where otherwise noted, all analyses were performed with software of the R project for statistical computing, version 3.3.0 (Bioconductor).
  • Bone marrow was flushed from the femur and tibia from C57BL/6 mice and was used without any digestion.
  • BM cells were incubated in 10 mL of RPMI 1640 medium with 10% heat-inactivated fetal calf serum (Biochrom, Cambridge, UK), penicillin/streptomycin, and 50 mM ⁇ -mercaptoethanol with FLT3L (200 ng/ml) combined with GM-CSF (5 ng/ml) for 9 days. Cells were then replated with the same combination of cytokines, and collected at day 15.
  • RNA 100 ng was assayed on a nCounter Digital Analyzer (NanoString) according to the manufacturer's instructions. Data were normalized by scaling with the geometric mean of the built-in control gene probes for each sample. Then, a log transformation (base 2) was performed. The heat maps showing levels of gene expression for differentially-expressed genes between neutrophils infiltrating the skin in the absence or presence of cDC1 were plotted and analysed using the GENE-E software, version 3.0.230 (Broad Institute, Cambridge, MA)
  • the inventors used Lysozyme M-GFP/ langerin-DTR albino mice for all intravital imaging experiments, wherein all the cells expressing lysozyme (neutrophils) are labeled green [ Li, J. L., C. C. Goh, et al. (2012). "Intravital multiphoton imaging of immune responses in the mouse ear skin.” Nat Protoc 7(2): 221-234 ; and Scheiermann, C., P. S. Frenette, et al. (2015). "Regulation of leucocyte homeostasis in the circulation.” Cardiovasc Res 107(3): 340-351 ]. Imaging of the ear was performed as previously described (Li at al 2012).
  • mice were anaesthetized with a cocktail of ketamine and xylazine (10 ⁇ l per gr of body weight) and then placed onto a heat pad to maintain body temperature at 37°C throughout the imaging procedure.
  • Depilatory cream was applied for 2 min to remove hair on the upper two-thirds of the dorsal side of the mouse ear.
  • the ear was then mounted on a custom-made ear skin stage for intravital multiphoton imaging.
  • Data correction and analysis were conducted using Imaris software (Bitplane, South Windsor, CT). Multiphoton mosaic imaging was performed to capture the entire volume of the granuloma and its surrounding interstitium.
  • the granuloma was quantified on Imaris using the surfacing tool, with a voxel size of 1 um. The percentage of granuloma was calculated (total volume of granuloma/total imaged volume *100; where total imaged volume was normalized across experiments).
  • Intra-dermal injection of P. acnes causes ear swelling, successive waves of immune cell infiltration, and fibrosis
  • CFU colony forming units
  • the inventors performed a comprehensive high-dimensional analysis of the ear cell populations using mass Cytometry by Time Of Flight (CyTOF) (Spitzer and Nolan, 2016, Ornatsky et al., 2006, Bendall et al., 2011, Tanner et al., 2013, Becher et al., 2014).
  • Cell suspensions were generated (Ginhoux et al., 2007) from P. acnes- and PBS- injected ears at 12 hours, and 2 and 5 days post-injection.
  • the inventors then labeled the cells with a panel of antibodies recognizing 37 different molecules ( Table 1 ) to allow the inventors to identify the different myeloid and granulocytic populations present (figure not shown).
  • the CyTOF data were analyzed using One-SENSE (One-dimensional Soli-Expression by Nonlinear Stochastic Embedding) (Cheng et al., 2015), a new dimensionality-reduction method based on t-stochastic neighbor embedding (tSNE), which enables unbiased visualization of high-dimensional similarities of cells in a two-dimensional map.
  • One-SENSE One-dimensional Soli-Expression by Nonlinear Stochastic Embedding
  • tSNE t-stochastic neighbor embedding
  • One-SENSE allows to first define expression of lineage-imprinted markers as the first dimension, thereby grouping broadly-ontogenetically-related cells together, and then to allow the other markers to define the second dimension, thus revealing any heterogeneity within the main clusters (figure not shown), as recently published for the analysis of murine tissue DC composition (Guilliams et al., 2016).
  • the inventors defined the "lineage dimension" using 5 markers that are differentially-expressed by immune cell populations: Ly6G for neutrophils, Ly6C for monocytes, CD11b and MHC-II for DC, and CD64 for macrophages (figure not shown).
  • the CyTOF data revealed within the Ly6G - cell population (figure not shown), the presence of LC (CD11b int MHCII hi CD64 neg CD24 hi ), cDC1 (CD11b neg MHCII hi CD64 neg CD24 hi ), cDC2 (CD11b hi MHCII hi CD64 neg CD172a hi ), CD11b - CD103 - double negative (DN) DC (CD11b neg MHCII hi CD24 neg CD64 neg ), mast cells (CD11b neg MHCII neg CD64 neg FC ⁇ RI int ), Ly6C lo monocytes (CD11b hi MHCII neg CD64 neg Ly6C neg/low ), Ly6C hi monocytes (CD11b hi MHCII neg CD64 neg Ly6c hi CCR2 hi ) and macrophages (CD11b hi MHCII neg CD64 hi Ly6C neg ).
  • CD11b hi MHCII hi CD64 int-hi Ly6C low FC ⁇ RI lo-int cells were classified as monocyte-derived inflammatory cells (MC), with phenotypic features of both macrophages and DC (Guilliams et al., 2014, Ginhoux et al., 2012, Malissen et al., 2014), but distinguishable by their expression of CD64 and low-intermediate levels of Ly6C (figure not shown), coupled with lack of expression of the DC lineage marker, zinc finger transcription factor Zbtb46 (Btbd4) (figure not shown) (Satpathy et al., 2012a, Satpathy et al., 2012b, Meredith et al., 2012).
  • MC monocyte-derived inflammatory cells
  • the inventors then confirmed the presence and absolute abundance of these populations in skin cell suspensions during the first 30 days post-injection with P. acnes using conventional flow cytometry (figure not shown). From these data the inventors can see that four distinct waves of immune cells pass through P .
  • acnes -injected skin from 8 hours, neutrophils, which peak around the end of day 1 and decline to disappearance around day 9; then Ly-6C high monocytes which although peaking at day 1 accumulate at high level from day 4 to day 9 declining between days 9 and 15 post-infection; while MC accumulate from day 5, peaking at day 9, consistent with their likely derivation from the earlier-infiltrating monocytes and finally macrophages accumulating slowly overtime until day 9 (figure not shown); lastly, T lymphocytes infiltrate the skin from day 5 and remain abundant until day 15 (figure not shown).
  • cDC1 are required for sustained immune cell recruitment following intra-dermal injection of P. acnes
  • D2 the mice lacking both cDC1 and LC
  • LG-DTR mice 24h post-DT exhibited significantly less ear swelling than did WT B6 mice
  • D9 P. acnes WT B6 mice versus D9 P. acnes Langerin DTR mice 24h post-DT-injection: 0.8 ⁇ 0.08 mm vs 0.373 ⁇ 0.02 mm; p 0.0.0022) (data not shown).
  • LC and/or cDC1 seem to be required for the progressive inflammatory response to intra-dermal P. acnes injection.
  • the inventors then asked how the depletion of cDC1 and LC affected the abundance of other immune cell types in the ears of P. acnes-injected mice.
  • the inventors observed similar numbers of neutrophils, macrophages and Ly-6C high monocytes in the first 18 hours after P. acnes infection in the presence or absence of both cDC1 and LC (data not shown).
  • the absence of langerin + cells resulted in significant decreases in the number of neutrophils, macrophages and Ly-6C high monocytes compared to WT mice (data not shown).
  • mice To distinguish the roles of LC and cDC1 in this phenomenon, the inventors exploited the differential repopulation characteristics of the two cell populations in langerin-DTR mice. The inventors compared mice injected with DT 24 hours before P. acnes challenge, depleted of both LC and cDC1, to mice injected with DT 30 days before, in which LC remain absent while cCD1 have fully repopulated the dermis (Ginhoux et al., 2007) (data not shown). In the presence of cDC1 alone, inflammatory ear thickening in response to P . acnes injection was comparable to WT mice, again peaking around day 9 (D9 P. acnes WT B6 mice versus D9 P.
  • the inventors compared the gene expression profiles of neutrophils from the skin of non-depleted LG-DTR mice (control mice) and LG-DTR mice 24 hours post-DT-injection (depleted mice).
  • the inventors identified 297 (178 down-regulated and 119 up-regulated), 149 (78 down-regulated and 71 up-regulated), and 227 (103 down-regulated and 124 up-regulated) differentially-expressed genes (DEG) between neutrophils from control and cDC1-depleted mice at 12 hours, and 2 and 4 days post-infection, respectively ( Table 2 ).
  • IPA Ingenuity Pathway Analysis
  • IPA showed that most of the GO biological processes that the DEGs were enriched for were related to neutrophil immune function, and specifically to their recruitment to inflamed sites (data not shown). Some of the GO biological processes were also related to monocyte recruitment, such as Colony Stimulating Factor 1 (CSF-1) (data not shown). Down-regulation of neutrophil CSF-1 expression likely explains the decreased monocyte recruitment observed in the absence of cDC1, as neutrophil-derived CSF-1 controls monocyte influx into inflamed tissue (Wang et al., 2016).
  • CSF-1 Colony Stimulating Factor 1
  • the inventors next used intravital multiphoton microscopy to ask whether these changes in neutrophil gene expression in the absence of cDC1 had functional correlates in vivo during the early stages of P. acnes infection.
  • the inventors employed albino Lysozyme M-GFP/Langerin-DTR mice, in which all cells expressing lysozyme (neutrophils) are labeled in green (Li et al., 2012) and Langerin-expressing LC and cDC can be depleted by DT administration.
  • non-depleted control mice one day after P. acnes injection, neutrophils swarmed in a coordinated fashion, moving rapidly in the extravascular space and accumulating around the P.
  • RIPK1 Receptor-Interacting Protein Kinase 1
  • MLKL Mixed Lineage Kinase domain-Like
  • NETosis compromised the ability of neutrophils to generate NETs.
  • a prerequisite for NETosis is the modification of arginine residues to citrulline on histones by a specific enzyme (peptidyl arginine deiminase 4, PAD4), which leads to massive chromatin de-condensation (Wang et al., 2009).
  • Quantification of the ratio of histone H3 citrullinated (H3Cit) to normal histone H3 (H3) by Western blot revealed a significant increase in the H3Cit/H3 ratio in undepleted versus depleted mice.
  • MMP9 Matrix Metallopeptidase 9
  • MMP9 KO mice (Vu et al., 1998) exhibited reduced ear thickness and a marked impairment of neutrophil, macrophage and Ly-6C high monocyte recruitment after P. acnes infection (data not shown).
  • the inventors also identified a down-regulation of MMP2 expression at the gene expression level in absence of cDC1 (data not shown), a Matrix Metallopeptidase known to promote early neutrophil recruitment in association with MMP9 (Song et al., 2015).
  • Neutrophils are short-lived cells in the steady state, but during inflammation their lifespan increases through activation-induced inhibition of apoptosis (Costantini et al., 2010, Elbim et al., 2009). Thus the reduced expression of activation-related genes in neutrophils from LC and cDC1-depleted mice might also increase neutrophil apoptosis.
  • the inventors measured expression of the early apoptotic marker Annexin-V, and staining with the DNA dye DAPI (indicating dead cells) across the neutrophil populations from the ear skin of mice injected with P. acnes.
  • Multi-dimensional analyses are now revealing previously-unappreciated levels of functional heterogeneity within immune cell subsets (See et al., 2016), therefore the inventors applied single-cell mRNA sequencing to ask, at both the individual cell and population level, how dermal cDC1 might be mediating neutrophil recruitment and function.
  • the inventors first profiled the transcriptomic signature of 67 single dermal cDC1 isolated from the ears of WT mice, 16 hours after P. acnes injection, then used these data to group cells sharing distinct patterns of gene expression. Using the 67 single cell transcriptomes, the inventors performed Principal Component Analysis (PCA) (data not shown), followed by hierarchical clustering of the 400 most variably-expressed genes using Fluidigm SINGuLAR and SPIN (See Methods) (Szulwach et al., 2015, Tsafrir et al., 2005).
  • PCA Principal Component Analysis
  • the SINGuLAR approach revealed two distinct cell clusters within the 67 cell cDC1 population: a minor cluster of 8 cells (Subset 1) and a major cluster of 59 cells (Subset 2) (data not shown).
  • IPA revealed that most of the DEGs expressed more abundantly in the minor "Subset 1" were related to DC immune function after capture of antigen, activation, phagocytosis, priming and migration pathways; specifically genes related to DC migration to the lymph node, maturation and activation during immune response and also to antigen priming and presentation by MHC-I (data not shown).
  • the inventors then asked whether these cDC1 clusters could be retrospectively identified in skin based on differences in expression of membrane proteins encoded by DEGs highlighted in the single cell transcriptome dataset.
  • the inventors first confirmed by flow cytometry that the transcript abundance for the surface markers EpCAM, CD59 and Ly6D differed significantly in the two cDC1 subsets (data not shown).
  • the inventors used flow cytometry to measure the frequency and intensity of cell-surface protein level expression of these markers across the dermal cDC1 population from both inside and outside the granulomatous area of P. acnes -or PBS- injected skin. This confirmed the presence of the minor population of cDC1 expressing high levels of CD59, EpCAM and Ly6D (i.e.
  • the EpCAM + CD59 + Ly6D + cDC1 subset was enriched inside the granuloma (data not shown), suggesting a link between the presence of the bacteria and the increased expression of these markers by cDC1.
  • the inventors confirmed that these markers were specific to cDC1 and not shared by cDC2 (data not shown).
  • an activated dermal cDC1 subset (“the EpCAM + CD59 + Ly6D + cDC1 subset”) can be distinguished within the cDC1 population at both the transcriptional and surface marker level.
  • Activated dermal cDC1 express neutrophil chemo-attractant VEGF ⁇ in the presence of P. acnes
  • the inventors then screened the single-cell cDC1 gene expression dataset for secreted molecules that could play a role in neutrophil recruitment and activation. Multiple potential neutrophil-modulatory candidates were expressed at a significantly higher level in the activated minor cDC1 subset, including cxcl1, il22, il23 ⁇ , il19, il7, il1 ⁇ and vegf ⁇ (data not shown).
  • VEGF/VEGFR regulate neutrophil infiltration into tissues during infection (Christoffersson et al., 2012, Massena et al., 2015)
  • the inventors asked whether cDC1 produced this protein in response to P. acnes.
  • Bone marrow-derived cDC1 produced increasing amounts of VEGF ⁇ in response to escalating densities of the bacterium and to similar levels than after Toll-like receptor (TLR) agonists, except the agonist of TLR7/8 (CLO75) (data not shown).
  • TLR Toll-like receptor
  • the inventors tested the expression profile of VEGF receptors and other markers on neutrophils infiltrating the skin in the presence or absence of cDC1. Using the neutrophil-specific CyTOF panel ( Table 6 ), the inventors uncovered marked changes in bone marrow, blood and skin neutrophil phenotype in response to P. acnes.
  • Table 6 Table of antibodies used for mass cytometry analysis listing metal conjugate, antibody clone name, supplier Isotope tag Sequence Clone/Company Qdot (Cd112/114) CD19 6D5 (Invitrogen) Pd--102 Barcode Pd--104 Barcode Pd---105 Barcode Pd--106 Barcode Pd--108 Barcode Pd--110 Barcode Ln--113 Barcode Ln--115 CD90 53-2.1 (Biolegend) La--139 CD45 30--F11 (Biolegend) Nd--141 CD43 eBioR2/60 (Ebioscience) Nd--142 MHC class II Y3P (BioXcell) Nd--143 CD49D R1-2 (Ebioscience) Nd--144 CD11a FD441.8 (BD) Nd--145 CD64 X54-5/7.1 (Biolegend) Sm--147 Ly6G IA8 (BD) Nd--148 Ly6c HK1.4 (Biolegend) N
  • Bone marrow neutrophils upregulated overtime CXCR2 and CD62L expression, two markers known to regulate the neutrophil release from bone marrow to the blood circulation (Eash et al., 2010, Martin et al., 2003). Blood neutrophils expressed high levels of CXCR2 associated with an upregulation of the expression of CD62L and CD49d, two markers associated to the tethering and rolling along postcapillary venules, prior to transmigration across the vascular endothelium to sites of inflammation (Rogowski et al., 1998, Furze and Rankin, 2008, Scheiermann et al., 2015) (data not shown).
  • cDC1-derived VEGF ⁇ is necessary for the inflammatory response to P. acnes
  • the transcription factors CREB, STAT3 and HIF- ⁇ regulate VEGF ⁇ expression by in vitro generated DCs (Salvi et al., 2016).
  • the inventors used a Cre/flox strategy in which HIF- ⁇ expression was specifically absent in cDC.

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US11382955B2 (en) 2019-11-25 2022-07-12 The Regents Of The University Of California Long-acting VEGF inhibitors for intraocular neovascularization
US11524053B2 (en) 2018-01-26 2022-12-13 The Regents Of The University Of California Methods and compositions for treatment of angiogenic disorders using anti-VEGF agents

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Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999032619A1 (en) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Genetic inhibition by double-stranded rna
US5981732A (en) 1998-12-04 1999-11-09 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-13 expression
US6046321A (en) 1999-04-09 2000-04-04 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-i1 expression
US6107091A (en) 1998-12-03 2000-08-22 Isis Pharmaceuticals Inc. Antisense inhibition of G-alpha-16 expression
WO2001036646A1 (en) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibiting gene expression with dsrna
WO2001068836A2 (en) 2000-03-16 2001-09-20 Genetica, Inc. Methods and compositions for rna interference
US6365354B1 (en) 2000-07-31 2002-04-02 Isis Pharmaceuticals, Inc. Antisense modulation of lysophospholipase I expression
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
WO2003017917A2 (de) * 2001-08-23 2003-03-06 Rina Netzwerk Rna-Technologien Gmbh Verwendung von molekularbiologisch hergestellten, nicht-viralen wirkstoffen zur behandlung der akne
US6566135B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 6 expression
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
WO2017062837A1 (en) * 2015-10-08 2017-04-13 Amd Therapeutics Llc Treatment of skin disorders by topical administration of vegf inhibitors

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999032619A1 (en) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Genetic inhibition by double-stranded rna
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
US6107091A (en) 1998-12-03 2000-08-22 Isis Pharmaceuticals Inc. Antisense inhibition of G-alpha-16 expression
US5981732A (en) 1998-12-04 1999-11-09 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-13 expression
US6046321A (en) 1999-04-09 2000-04-04 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-i1 expression
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
WO2001036646A1 (en) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibiting gene expression with dsrna
WO2001068836A2 (en) 2000-03-16 2001-09-20 Genetica, Inc. Methods and compositions for rna interference
US6365354B1 (en) 2000-07-31 2002-04-02 Isis Pharmaceuticals, Inc. Antisense modulation of lysophospholipase I expression
US6566135B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 6 expression
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
WO2003017917A2 (de) * 2001-08-23 2003-03-06 Rina Netzwerk Rna-Technologien Gmbh Verwendung von molekularbiologisch hergestellten, nicht-viralen wirkstoffen zur behandlung der akne
WO2017062837A1 (en) * 2015-10-08 2017-04-13 Amd Therapeutics Llc Treatment of skin disorders by topical administration of vegf inhibitors

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHENG, Y.; M. T. WONG ET AL.: "Categorical Analysis of Human T Cell Heterogeneity with One-Dimensional Soli-Expression by Nonlinear Stochastic Embedding", J IMMUNOL, vol. 196, no. 2, 2016, pages 924 - 932
GINHOUX, F.; M. P. COLLIN ET AL.: "Blood-derived dermal langerin+ dendritic cells survey the skin in the steady state", J EXP MED, vol. 204, no. 13, 2007, pages 3133 - 3146
GUILLIAMS, M.; C. A. DUTERTRE ET AL.: "Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species", IMMUNITY, vol. 45, no. 3, 2016, pages 669 - 684, XP029755883, DOI: doi:10.1016/j.immuni.2016.08.015
LI, J. L.; C. C. GOH ET AL.: "Intravital multiphoton imaging of immune responses in the mouse ear skin", NAT PROTOC, vol. 7, no. 2, 2012, pages 221 - 234
SCHEIERMANN, C.; P. S. FRENETTE ET AL.: "Regulation of leucocyte homeostasis in the circulation", CARDIOVASC RES, vol. 107, no. 3, 2015, pages 340 - 351
ZHANG GUO-YOU ET AL: "Inhibition of vascular endothelial growth factor expression in keloid fibroblasts by vector-mediated vascular endothelial growth factor shRNA: a therapeutic potential strategy for keloid", ARCHIVES OF DERMATOLOGICAL RESEARCH, SPRINGER, INTERNATIONAL, BERLIN, DE, vol. 300, no. 4, 31 March 2008 (2008-03-31), pages 177 - 184, XP009501806, ISSN: 0340-3696, DOI: 10.1007/S00403-007-0825-Y *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11524053B2 (en) 2018-01-26 2022-12-13 The Regents Of The University Of California Methods and compositions for treatment of angiogenic disorders using anti-VEGF agents
US11382955B2 (en) 2019-11-25 2022-07-12 The Regents Of The University Of California Long-acting VEGF inhibitors for intraocular neovascularization
US11433118B2 (en) 2019-11-25 2022-09-06 The Regents Of The University Of California Long-acting VEGF inhibitors for intraocular neovascularization
US11576948B2 (en) 2019-11-25 2023-02-14 The Regents Of The University Of California Long-acting VEGF inhibitors for intraocular neovascularization

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